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Adams Hawii 0085O 10232.Pdf ANALYSIS AND DEVELOPMENT OF MANAGEMENT TOOLS FOR ORYCTES RHINOCEROS (COLEOPTERA: SCARABAEIDAE) A THESIS SUBMITTED TO THE GRAUDATE DIVISION OF THE UNIVERSITY OF HAWAIʻI AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN TROPICAL PLANT PATHOLOGY MAY 2019 By Brandi-Leigh H. Adams Thesis Committee: Michael Melzer, Chairperson Zhiqiang Cheng Brent Sipes ACKNOWLEDGEMENTS It is with deep gratitude that I thank the members of my committee, Dr. Michael Melzer, Dr. Zhiqiang Cheng, and Dr. Brent Sipes for their expert advice and knowledge, to which I have constantly deferred to during my time as a graduate student. A very special thank you goes to Dr. Michael Melzer, who took me in as an undergraduate lab assistant, and saw enough potential in me that he felt I deserved the opportunity to learn, travel, and grow under his guidance. I would also like to give special thanks to Dr. Shizu Watanabe, who always made time to answer even the smallest, most O.C.D. of my questions, who gave me words of encouragement when experiments did not go as planned or when I would find myself in doubt, and who has become a mentor and friend along the way. To my very first mentors in science; Dr. Wendy Kuntz, Dr. Matthew Tuthill, and Keolani Noa; thank you for encouraging me to pursue a major and career in STEM in the first place. I would also like to thank my lab mates, Nelson Masang Jr., Alexandra Kong, Alejandro Olmedo Velarde, Tomie Vowell, Asoka De Silva, Megan Manley, Jarin Loristo, and Cheyenne Barela for their support with experiments, and the knowledge and skills they have passed on to me. My gratitude also goes out to my fellow graduate students, who have learned, grown, and triumphed alongside me. To all of the wonderful people in the PEPS department, I thank you for being my educators, and for always being kind and supportive. I would also like to thank the following collaborators for their help in acquiring the samples needed to conduct this research, and for their generosity in sharing their own collection of samples: the HDOA CRB Response Team, Dr. Keith Weiser, Darcy Oishi, Dr. Joel Miles, Dr. Christopher Kitalong, the faculty and students of Palau Community College, Dr. Daniel Rubinoff, J. Bradley Reil, Dr. Aubrey Moore, Roland Quitugua, Sami Soni, and Riten Gosai. ii Finally, from the bottom of my heart, I would like to thank my family and friends. To my mother, Tracy, I would like to thank for her eternal love and support. Thank you for all the advice and knowledge that has helped me grow as a person with each passing year. I hope I have made you proud. To my best friend, Kaliko, thank you for your love and understanding, for being the rock to my ʻopihi all these years, and for helping me see this through to the end. You inspire me endlessly to do better and live fully. To my grandmother, Cicinia and my brother, Brandon, thank you for being your crazy selves, and for adding laughter to my life when I need it most. To Mark, thank you for being an inquisitive mind and for taking a genuine interest in my research. To Eleanor, Alfred, and Norma, my second family, thank you for opening your home and your hearts to me, and making me feel like part of the family. To my precious fur babies, thank you for being a constant source of love and happiness, especially Kahu and Kui, who are no longer here to walk with me to the finish line, but were there from the start to comfort me during the hardest of times. Success is not won without the help of others, and I am eternally grateful to have had all of you supporting me throughout this journey. The culmination of this research is a result of hard work and dedication, not just of my own volition, but of all of yours’ too. It is my hope that this body of work is something we can all be proud of. “If I have seen further it is by standing on the shoulders of giants.” -Sir Isaac Newton iii ABSTRACT Oryctes rhinoceros (Coleoptera: Scarabaeidae) is a pestiferous beetle causing severe damage to coconut and other palms by boring into and feeding upon the crown tissue. Several tools are used to manage this beetle, including pheromone lured traps and the biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). The pheromone lure, ethyl 4-methyloctanoate, was assessed for its attractive ability towards the CRB-S and CRB-G haplotypes by comparing the distribution of the haplotypes in non trap-caught and trap-caught populations collected from Palau. A diagnostic qPCR assay was designed to detect OrNV, and was found to be a more sensitive and accurate method for OrNV detection compared to the conventional PCR assay. Finally, because the newly emerged CRB-G haplotype has acquired resistance to OrNV, RNA extractions were sent for high-throughput sequencing to discover other viruses present in O. rhinoceros. Three sequences were discovered to be similar in sequence to viruses of other insect species. iv TABLE OF CONTENTS Acknowledgements…………………………………………………………………………….....ii Abstract…………………………………………………………………………………………...iv List of Tables…………………………………………………………………………………......ix List of Figures…………………………………………………………………………………......x Chapter 1: Literature Review………………………………………………………………….......1 The Coconut Rhinoceros Beetle………………………………………………………......1 Impacts of Oryctes rhinoceros Damage………………………………………………......2 Control and Management Practices…………………………………………………….....4 Oryctes rhinoceros nudivirus as a Biological Control……………………………….........6 A New Oryctes rhinoceros Haplotype Resistant to OrNV……………………………......8 Significance of Oryctes rhinoceros in Hawaiʻi……………………………………….......9 Chapter 2: Assessment of Attraction to Ethyl 4-methyloctanoate Male Aggregation Pheromone Between Two Oryctes rhinoceros Haplotypes in Palau…………………………….11 Introduction………………………………………………………………………………11 Materials and Methods…………………………………………………………………...12 Trapping Oryctes rhinoceros Specimens………………………………………...12 Sample Collection………………………………………………………………..12 Sample Selection………………………………………………………………....14 DNA Extraction from Oryctes rhinoceros Specimens………………………......14 Genotyping Oryctes rhinoceros Specimens……………………………………...14 Data Analysis…………………………………………………………………….16 Results…………………………………………………………………………………....16 v Discussion……………………………………………………………………………......23 Chapter 3: Development of a Multiplex Quantitative Polymerase Chain Reaction Assay for Rapid and Sensitive Detection of the Oryctes rhinoceros nudivirus………………....28 Introduction……………………………………………………………………………....28 Materials and Methods……………………………………………………………….......30 Sample Collection………………………………………………………………..30 Total DNA Extraction…………………………………………………………....30 Primer Selection and Optimization………………………………………………30 Arginine Kinase Degenerate Primer Set PCR Optimization…………….31 RNA pol II Degenerate Primer Set PCR Optimization………………….33 Sequencing Arginine Kinase and RNA pol II PCR Products…………………....33 Ligation of AK or PL PCR Products to Promega p-GEM®-T Easy Vector System I………………………………………………….....34 Transformation of Ligated AK or PL PCR Products Into DH5α Escherichia coli Cells……………………………………….........34 Screening Transformed DH5α Escherichia coli Cells…………………...34 Plasmid Extraction……………………………………………………….35 Preparing Plasmid Extracts for Sequencing……………………………...35 Sequence Analysis……………………………………………………………….36 Designing Primer-Probe Sets for Arginine Kinase, RNA pol II, and OrNV………………………………………………………………………...38 qPCR Assay Development……………………………………………………….38 qPCR Assay Efficiency Trials…………………………………………………...40 vi qPCR Test Designs………………………………………………………………41 Data Analyses…………………………………………………………………....42 Results……………………………………………………………………………………44 OrNV Multiplex qPCR Assay Validation………………………………………..44 Detecting Strains of OrNV……………………………………………………….44 Comparing the PCR and qPCR Assays for Detection of OrNV…………………44 Determining the Status of OrNV in Hawaiʻi’s Oryctes rhinoceros Population……………………………………………………………50 Relative Quantification of OrNV in Oryctes rhinoceros Tissues………………..50 Discussion………………………………………………………………………………..50 Chapter 4: Virus Discovery in Oryctes rhinoceros Via High-throughput Sequencing Technology………………………………………………………………………….59 Introduction………………………………………………………………………………59 Materials and Methods…………………………………………………………………...60 Total and Small RNA Extraction for Illumina MiSeq…………………………...60 Sample Selection………………………………………………………....60 Total and Small RNA Extraction…………………………………...........61 dsRNA Extraction for Illumina MiSeq………………………………………......62 Sample Selection………………………………………………………....62 dsRNA Extraction……………………………………………………......62 cDNA Library Synthesis………………………………………………....63 Screening cDNA Products…………………………………………….....63 Preparing Samples for Illumina MiSeq…………………………………..63 vii Data Analysis…………………………………………………………….............64 Results……………………………………………………………………………………65 Illumina MiSeq Results from Total and Small RNA Extractions………………..65 Illumina MiSeq Results from dsRNA Extraction………………………………..67 Discussion………………………………………………………………………………..73 Chapter 5: Conclusion and Future Studies…………………………………………………….....81 Literature Cited………………………………………………………………………………......84 viii LIST OF TABLES Table Page 1. Number of non trap-caught and trap-caught Oryctes rhinoceros samples collected per state in Palau………………………………………………………………13 2. Degenerate primer sets designed for amplification of nuclear protein- coding genes in Coleopteran species………………………………………………….....32 3. Consensus sequences of the Oryctes rhinoceros arginine kinase and RNA polymerase II nuclear genes, and the OrNV glycoprotein gene………………………....37 4. TaqMan primer-probe sets for arginine kinase,
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